This study, we sought to extend these in Bay 43-9006 Nexavar vitro findings to an in vivo setting to explore the anti tumor potential of GSK 3b inhibitors in prostate cancer. Using mouse xenograft model and a spontaneous mouse prostate cancer model, we demonstrated that GSK 3 inhibitors significantly suppressed tumor development and growth. We also documented that CCAAT/ enhancer binding protein alpha is accumulated in GSK 3 inhibitor treated prostate cancer cells, which might represent a mechanism involved in GSK 3 inhibitor induced anti tumor effect. MATERIALS ANDMETHODS Cell Culture, Antibodies, and Reagents PC 3 and C4 2 cells were maintained in a humidified atmosphere of 5% CO2, RPMI 1640 supplemented with 10% fetal bovine serum plus antibiotics. TDZD 8 was obtained from Calbiochem. L803 mts was synthesized by GeneMed Synthesis as previously described. Antibodies for C/EBPa,C/EBPb, Ki 67, and Actin were purchased from Santa Cruz Biotech. Lithium chloride andother chemicals were purchased from Sigma. Where indicated the inhibitor was added to the cell culture media from a 1,000 fold concentrated stock. Control cultures received similar amounts of the solvent only. Final concentrations of the solvent did not exceed 0.1%. Animal Experiments and Immunohistochemistry All animal studies were conducted under an approved Institutional Animal Care and Use Committee protocol. For xenograft experiments in nude mice, PC 3 or C4 2 cell suspension were inoculated subcutaneously into the rear flanks of 6 weeks old male nude mice. For the protocol A of LiCl treatment, animals were randomly divided into two groups on next day after PC 3 cell inoculation. One group received intraperitoneal LiCl injection at a daily dose of 2.0 mg/ kg bodyweight. LiCl was dissolved in PBS. Control animals received PBS alone in the same volume. Xenograft development and growth were recorded by measuring tumor dimensions twice a week with a caliper. Tumor volume was calculated by the formula of LWH0.5236. Treatment lasted for 8 weeks.
At the end of treatment, tumors were extirpated, and the final tumor size and weight, as well as animal weight were measured and recorded. For the protocol B, treatment began once xenograft tumors became palpable or 30mm3 in size, LiCl or PBS injection was conducted as above for a 2 week course. Tumor size and wet weight weremeasured at necropsy. For TDZD 8 treatment, when Imatinib CGP-57148B PC 3 or C4 2 xenografts were in size of 30mm3, animals were randomized into two groups. TDZD 8 was dissolved in a slow releasing formula containing 50% N,Ndimethylacetamide, 45% polyethylene glycol 400, and 5% Tween 80. Drugs or the solvent in 100 ml volume was injected intraperitoneally three times a week. TDZD 8 was used at a dose of 4.0 mg/kg bodyweight per injection. Tumor growth and measurement were monitored as above. For the TRAMP model system, C57BL/6 TRAMP mice were obtained fromNCI FrederickMouseModels of Human Cancer Consortium Repository and maintained as instructed. Male transgenic mice at age of 10 weeks were used in the experiments. TDZD 8, L803 mts, or the solvent in 100 ml volume was injected i.p. three times a week for a period of 4 weeks. L803 mts were dissolved in the same slow releasing solvent as TDZD 8. At the end of experiments, necropsy was performed on all animals.